N-Acetylcysteine Decreases Myocardial Content of Inflammatory Mediators Preventing the Development of Inflammation State and Oxidative Stress in Rats Subjected to a High-Fat Diet

Arachidonic acid (AA) is a key precursor for proinflammatory and anti-inflammatory derivatives that regulate the inflammatory response. The modulation of AA metabolism is a target for searching a therapeutic agent with potent anti-inflammatory action in cardiovascular disorders. Therefore, our study aims to determine the potential preventive impact of N-acetylcysteine (NAC) supplementation on myocardial inflammation and the occurrence of oxidative stress in obesity induced by high-fat feeding. The experiment was conducted for eight weeks on male Wistar rats fed a standard chow or a high-fat diet (HFD) with intragastric NAC supplementation. The Gas-Liquid Chromatography (GLC) method was used to quantify the plasma and myocardial AA levels in the selected lipid fraction. The expression of proteins included in the inflammation pathway was measured by the Western blot technique. The concentrations of arachidonic acid derivatives, cytokines and chemokines, and oxidative stress parameters were determined by the ELISA, colorimetric, and multiplex immunoassay kits. We established that in the left ventricle tissue NAC reduced AA concentration, especially in the phospholipid fraction. NAC administration ameliorated the COX-2 and 5-LOX expression, leading to a decrease in the PGE2 and LTC4 contents, respectively, and augmented the 12/15-LOX expression, increasing the LXA4 content. In obese rats, NAC ameliorated NF-κB expression, inhibiting the secretion of proinflammatory cytokines. NAC also affected the antioxidant levels in HFD rats through an increase in GSH and CAT contents with a simultaneous decrease in the levels of 4-HNE and MDA. We concluded that NAC treatment weakens the NF-κB signaling pathway, limiting the development of myocardial low-grade inflammation, and increasing the antioxidant content that may protect against the development of oxidative stress in rats with obesity induced by an HFD.


Introduction
N-acetyl-cysteine or N-acetylcysteine (NAC) is a wideknown drug acknowledged by the World Health Organization (WHO) as an essential medication. It is commonly used to treat paracetamol overdose, likewise as a mucolytic agent in certain respiratory diseases [1]. In many countries, it is available as an over-the-counter drug or nutraceutical, known for its antioxidative properties. Firstly, this acetylated precursor of L-cysteine has a direct antioxidant impact due to the free thiol group property-counteracting reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well. Indirectly, by upregulating the intracellular cysteine level NAC increases the rate of reduced glutathione (GSH) synthesis, the most common cellular antioxidant. Additionally, it possesses signifcant anti-infammatory properties as a result of nuclear factor κ B (NF-κB) suppression followed by diminished production of proinfammatory cytokines, i.e., tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), and interleukin 6 (IL-6) [1]. Some studies have shown the efect of NAC on the modulation of arachidonic acid (AA) metabolism as the main infammatory lipid precursor, which belongs to n − 6 polyunsaturated fatty acids (PUFA). Te efect of NAC on AA metabolism and prostaglandin formation has been demonstrated in activated monocytes and neurons after nerve tissue injury [1,2]. Te administration of NAC together with specifc and nonspecifc cyclooxygenase (COX) inhibitors-rofecoxib and diclofenac-signifcantly reduced the formation of prostaglandin E2 (PGE2) induced by lipopolysaccharides, enhancing the action of the above-mentioned COX inhibitors [2].
Given its properties, NAC is intensively studied in various clinical studies covering chronic metabolic disorders, including cardiovascular diseases (CVD), metabolic syndrome, liver complications, and psychiatric illnesses, in which oxidative stress and infammation are considered as risk factors for the development of mentioned conditions [3,4]. It is not debatable that the pandemic of obesity and other metabolic syndrome components has a huge impact on chronic cardiovascular diseases, which possess a relevant, growing problem in the global healthcare system [5]. Te chronic enhanced availability and infux of fatty acids (FA) favors ectopic lipid accumulation in peripheral tissues such as the liver and kidneys [6][7][8]. Consequently, an increase in lipid storage intensifes the low-grade infammation, the impairment in mitochondrial functioning, and oxidative stress development in the cardiac tissue favoring myocardial cell death and heart failure [9,10]. So, it is important to search for a therapeutic agent that will reveal new potentially anti-infammatory and antioxidative properties to improve cardiac functioning. Several clinical trials and animal studies have shown that NAC exerts noteworthy actions in cardiovascular disorders, particularly in acute myocardial ischemia and acute myocardial infarction, although its role in chronic cardiovascular diseases is still not fully understood [11,12]. Tus, our study aims to determine the potential protective impact of N-acetylcysteine supplementation on the occurrence of myocardial infammation in rats with obesity induced by a high-fat diet (HFD). In this sense, we will examine how NAC can alter the infammatory response by suppressing the activation of the arachidonic acid pathway. We will also explore the infuence of NAC on enzymatic and nonenzymatic antioxidant protection and the products of lipoperoxidation in cardiac injury induced by high-fat feeding.

Animals and Experimental
Protocol. Te experiment was conducted for eight weeks on male, four-week-old Wistar rats with an initial body weight of approximately 50-70 g. All animals were housed throughout the entire duration of the study in standard laboratory animal living conditions: plastic autoclavable cages, 22 ± 2°C air temperature, a 12 h reverse light/dark cycle, and unlimited access to water and standard rodent chow. After the frst week of adaptation to the new environment, the rats were randomly divided into four groups in equal numbers: 10 rats per experimental group; 40 rats for all groups in the experiment. Te characteristics of the groups were as follows: (1) control group-rats fed a standard rodent chow (65.5% calories from carbohydrate, 24.2% calories from protein, and 10.3% calories from fat; nutritional composition of the standard diet is presented in the Table 1; and Agropol, Motycz, and Poland); (2) the HFD group-rats fed a high-fat diet (59.8% calories from fat, 20.1% calories from protein, and 20.1% calories from carbohydrate; nutritional composition of the high-fat diet is shown in the Table 2; D12492, Research Diet, New Brunswick, NJ, USA); (3) NAC group-rats fed the abovedescribed standard diet plus N-acetylcysteine (Sigma-Aldrich, St. Louis, MO, USA); and (4) HFD + NAC group-rats fed a high-fat diet as well as N-acetylcysteine. Te experimental model of high-fat feeding was selected based on an accessible protocol to contribute to hyperlipidemic occurrence as a crucial factor for the development of obesity-related heart diseases [13][14][15]. Te NAC solution was administered to the appropriate groups once daily, between 8-9 am. Te substance was dissolved in a saline solution and immediately administered intragastrically by gastric gavage at a dose of 500 mg/kg of body weight. Te individuals from the control and HFD groups received only saline solution. Te amount of administered NAC was adjusted according to the current body weight of rats, and it was recalculated every two days. Te intragastric administration of NAC ensured that rats obtained the full dose appropriate for body weight. Te NAC dose was established based on available data to provide a satisfactory efect and eliminate the risk of toxicity in Wistar rats [16]. Te NAC solution was supplemented concomitantly with standard or high-fat diets to determine the potential preventive efect of NAC on cardiac lipid metabolism with particular reference to infammatory and oxidative alterations. At the end of the eight weeks, after a 12-hour overnight fast, the animals were anesthetized by intraperitoneal phenobarbital injection (80 mg/kg of body weight). Te left ventricle was excised and immediately frozen in liquid nitrogen using precooled aluminum thongs. Also, blood was collected in the tubes containing EDTA and then centrifuged to obtain plasma fractions. All samples (left ventricle tissue and plasma) were stored at −80°C until further measurements. Te study was approved by the Ethical Committee for Animal Testing in Bialystok (No. 21/2017).

Determination of the Myocardial and Plasma Arachidonic
Acid Concentration. Lipids obtained from the left ventricle and plasma samples were extracted using a solution of Agilent Technologies, Santa Clara, CA, USA) method was used to quantify the particular fatty acid methyl esters (FAME) level in each lipid fraction, depending on the retention times of the standard, as previously described by Chabowski et al. [18]. On the basis of the fatty acid composition in the selected lipid fractions, the concentration of arachidonic acid was estimated and expressed in nanomoles per milliliter of plasma or per gram of tissue.

Determination of the Myocardial Arachidonic Acid Derivatives and Oxidative Stress Parameters.
We applied the commercial enzyme-linked immunosorbent assay (ELISA) and the colorimetric assay kits to determine the concentration of arachidonic acid derivatives-prostaglandin E2  Before the determinations, the left ventricle tissue (25 mg) was homogenized in 1 ml of ice-cold phosphate bufer saline (PBS) for measurements of PGE2, PGI2, LTB4, LTC4, LXA4, SOD2, CAT, GSH, 4-HNE, and AGE, or in 250 μl of ice-cold RIPA bufer only for MDA testing. Te prepared homogenates suspended in PBS or RIPA bufer were centrifuged as reported by the manufacturer's protocols. After that, the supernatants were transferred into new tubes and frozen immediately at −80°C for analysis.
For the quantitative determinations, the absorbance of all parameters (except for the MDA determination) was detected spectrophotometrically at 450 nm on a microplate reader (Synergy H1 Hybrid Reader; BioTek Instruments, Winooski, VT, USA). Te calorimetric measurement at 530 nm was used to assess the content of MDA. Te concentration of the analyzed parameters was elaborated depending on the individual standard curves obtained for each measurement. Te results are expressed in millimoles (GSH), micromoles (MDA), nanograms (PGI2, LTC4, LXA4, CAT, AGE), or picograms (PGE2, LTB4, SOD2, 4-HNE) per milligram of tissue.

Determination of the Myocardial Anti-Infammatory and
Proinfammatory Cytokines and Chemokines. Te left ventricle lysates were prepared in cell lysis bufer (Bio-Rad, Hercules, CA, USA) with the addition of protease inhibitors-factor I and factor II (Bio-Rad, Hercules, CA, USA) and phenylmethylsulfonyl fuoride (PMSF; Sigma Aldrich, Saint Louis, MO, USA). Te lysates were centrifuged at 15, 000 × g for 10 min at 4°C. Subsequently, the obtained supernatants were transferred to new tubes and used to determine the total protein concentration. Te range of protein concentrations was 200-900 μg/ml.
2.6. Statistical Analysis. All data are expressed as the mean ± standard deviation (SD) based on ten independent determinations in each experimental group, except for the Western blot method, in which results are based on six independent determinations. Te statistical assessment was performed using a GraphPad Prism 8.2.1. (GraphPad Software; San Diego, CA, USA). Te normality of data distribution and homogeneity of the variance were assessed using the Shapiro-Wilk test and Bartlett's test. Te statistical comparisons were performed by a two-way ANOVA followed by a respective post hoc test (Tukey's test and t-test). A statistical signifcance was set at p < 0.05.

Efect of Eight-Week NAC Treatment on the Concentration of Arachidonic Acid in the Plasma of Rats Subjected to a Standard and a High-Fat Diets.
Our study revealed that an HFD caused a signifcant increase in plasma AA concentration in the PL fraction (HFD: +14.9%, p � 0.0393, vs. control group; Figure 1(a)). No obvious changes in the phospholipid's AA content were observed in rats treated with NAC (NAC: p � 0.5320, vs. control group, HFD + NAC: p � 0.6206 and p � 0.2595, vs. the control and HFD groups; Figure 1(a)). In the TAG fraction, NAC supplementation to rats fed an HFD resulted in a crucial reduction in the AA level (HFD + NAC: −34.6%, p � 0.0040; Figure 1(b)) compared to the HFD group, which is the objective manifestation of impaired infammation development. In relation to the standard condition, there were no signifcant changes in all examined groups (HFD: p � 0.4206, NAC: p � 0.8413, HFD + NAC: p � 0.0599; Figure 1(b)). Furthermore, in the DAG fraction, the AA content was noticeably increased in the HFD and HFD + NAC groups (HFD: +64.7%, p � 0.0035, HFD + NAC: +47.3%, p � 0.0129; Figure 1(c)) and noticeably decreased in the NAC alone group (NAC: −33.7%, p � 0.0002; Figure 1(c)) in relation to the control group. Plasma AA levels in the DAG pool did not substantially difer in the HFD with NAC treatment group (HFD + NAC: p � 0.2014; Figure 1(c)) compared to the HFD group. As excepted, the considerable increase in the content of AA in the FFA fraction caused by high-fat feeding (HFD: +21.0%, p � 0.0186, vs. control group; Figure 1

Efect of Eight-Week NAC Treatment on the Concentration of Arachidonic Acid in the Left Ventricle Tissue of Rats Subjected to a Standard and a High-Fat Diets.
In the lipid overload condition, the arachidonic acid content was considerably elevated in the PL fraction (HFD: +77.6%, p < 0.0001, vs. control group; Figure 2(a)). Concomitantly, there were appreciable changes in the myocardial AA content in the PL fraction in rats fed a high-fat diet with NAC supplementation (HFD + NAC: +60.4%, p < 0.0001 and −9.7%, p � 0.0159, vs. control and HFD groups, respectively; Figure 2(a)), which we can suppose that NAC supplementation has a preventive action by the reduction of cardiac AA content in the phospholipid pool. In comparison with the standard condition, we observed a pronounced rise in the arachidonic acid level in the TAG fraction in the HFD and HFD + NAC groups (HFD: +212.6%, p < 0.0001, HFD + NAC: +141.2%, p < 0.0001; Figure 2(b)). Moreover, NAC administration to rats receiving an HFD caused a signifcantly diminished the amount of AA in the TAG fraction (HFD + NAC: −22.8%, p � 0.0079, vs. HFD group; Figure 2(a)) for precautionary the onset of low-grade infammation. In the NAC alone group we observed no relevant alteration in the AA content in the PL and TAG fractions (PL − NAC: p � 0.7285; Figure 2(a); TAG − NAC: p � 0.8853; Figure 2(b)) in relation to the appropriate control group. Our study also revealed that the myocardial AA content in the DAG fraction was remarkably increased in the HFD group (HFD: +41.3%, p � 0.0007, vs. control group; Figure 2(c)). Additionally, in both NAC-treated groups we noticed a substantial decrease in the . Te arachidonic acid content in the selected lipid fractions was examined by the Gas-Liquid Chromatography (GLC) method. Te data are expressed as mean ± standard deviation (SD) and based on ten independent determinations (n � 10). Te signifcant diferences were assessed by two-way ANOVA followed by a respective post hoc test (Tukey's test and t-test). * p < 0.05, signifcant diference compared to the control group; # p < 0.05, signifcant diference compared to the HFD group.
In the HFD + NAC group we noticed no markedly elevation in the diacylglycerol's AA content (HFD + NAC: p � 0.2222; Figure 2(c)) compared to the standard condition. Te content of arachidonic acid in the FFA fraction was substantially enhanced in all examined groups (HFD: +40.2%, p < 0.0001, NAC: +6.7%, p � 0.0218, HFD + NAC: +14.3%, p � 0.0411; Figure 2(d)) in relation to the control group. We also observed a pronounced decrease in the AA level in the FFA fraction (HFD + NAC: −18.5%, p � 0.0009; Figure 2(d)) than the HFD group, so we can suppose that NAC has an anti-infammatory properties.

Efect of Eight-Week NAC Treatment on the Expression of Proteins from Eicosanoid Synthesis Pathway in the Left Ventricle Tissue of Rats Subjected to a Standard and a High-Fat
Diets. Under lipid overload condition, we noticed a significant increment in the total expression of COX-1 (HFD: +20.0%, p � 0.0022, vs. control group; Figure 3(a)), which was abolished by the chronic NAC administration (HFD + NAC: −16.5%, p � 0.0046, vs. HFD group; Figure 3(a)), thus revealing its preventive efect on infammation occurrence. Similar protective infuence was disclosed in the COX-2 expression (HFD: +24.4%, p � 0.0263, vs. Control group, HFD + NAC: −23.9%, p � 0.0020, vs. HFD group; Figure 3(b)). We also noticed a substantial increase in the expression of 5-LOX in rats fed a high-fat diet (HFD: +17.7%, p � 0.0252, vs. control group; Figure 3 . Te arachidonic acid content in the selected lipid fractions was examined by the Gas-Liquid Chromatography (GLC) method. Te data are expressed as the mean ± standard deviation (SD) and based on ten independent determinations (n � 10). Te signifcant diferences were assessed by two-way ANOVA followed by a respective post hoc test (Tukey's test and t-test). * p < 0.05, signifcant diference compared to the control group; # p < 0.05, signifcant diference compared to the HFD group.

Efect of Eight-Week NAC Treatment on the Concentration of Arachidonic Acid Derivatives in the Left Ventricle Tissue of Rats Subjected to a Standard and a High-Fat Diets.
In all examined groups the concentration of PGE2 was signifcantly elevated (HFD: +57.8%, p < 0.0001, NAC: +28.5%, p � 0.0306, HFD + NAC: +22.7%, p � 0.0424; Figure 4(a)) in relation to the standard condition. Moreover, NAC supplementation to rats fed an HFD provoked a signifcant reduction in the PGE2 amount (HFD + NAC: −22.3%, p � 0.0151; Figure 4(a)) compared to rats receiving a high- International Journal of Infammation 7 fat diet alone. We found that the concentration of PGI2 was no relevant in rats treated by HFD or/with NAC (HFD: p � 0.2213, NAC: p � 0.0703, HFD + NAC: p � 0.2978; Figure 4(b)) in comparison with the control group. In the HFD-induced obesity group, NAC treatment revealed a crucial increment in the PGI2 content (HFD + NAC: +26.0%, p � 0.0300, vs. HFD group; Figure 4(b)). As excepted, in the HFD group the concentration of LTB4 was greater (HFD: +15.9%, p � 0.0254; Figure 4(c)) than in the control group. After eight-week NAC supplementation to rats fed an HFD, the concentration of LTB4 was remarkably decreased (HFD + NAC: −11.7%, p � 0.0070, vs. HFD group; Figure 4(c)). In both NAC-treated group the LTB4 concentration was statistically unchanged (NAC: p � 0.3017, HFD + NAC: p � 0.6816, vs. control group; Figure 4(c)). In rats receiving a high-fat diet we noticed a signifcant increase in the LTC4 content (HFD: +8.2%, p � 0.0191, vs. control group; Figure 4 ). In rats treated by NAC application we found a markedly elevation in the level of LXA4 (NAC: +65.0%, p � 0.0037, vs. control group, HFD + NAC: +59.5%, p � 0.0007 and +130.0%, p < 0.0001, vs. control and HFD groups, respectively; Figure 4(e)). Treatment of NAC implies that obesity-induced infammation was decreased by the reduction of PGE2, LTB4 and LTC4 levels with simultaneously the elevation of PGI2 and LXA4 levels.

Efect of Eight-Week NAC Treatment on the Expression of Proteins Involved in the Infammatory Processes in the Left Ventricle Tissue of Rats Subjected to a Standard and a High-Fat
Diets. In the left ventricle homogenates, a high-fat diet induced a relevant reduction in the total expression of Nrf-2 (HFD: −24.1%, p � 0.0035; Figure 5(a)) in relation to the control group. In both NAC-treated groups we observed no signifcant increase in the Nrf-2 expression (NAC: p � 0.0617, vs. control group, HFD + NAC: p � 0.5949 and p � 0.1121, vs. control and HFD groups, respectively; Figure 5(a)). What is more, in rats fed a high-fat diet alone and a high-fat diet with NAC application the total expression of TGF-β was notably increased (HFD: +39.8%, p � 0.0135, HFD + NAC: +27.2%, p � 0.0314; Figure 5(b)) compared to . Te eicosanoids content was examined by the enzyme-linked immunosorbent assay (ELISA) kits. Te data are expressed as mean ± standard deviation (SD) and based on ten independent determinations (n � 10). Te signifcant diferences were assessed by two-way ANOVA followed by a respective post hoc test (Tukey's test and t-test). * p < 0.05, signifcant diference compared to the control group; # p < 0.05, signifcant diference compared to the HFD group. 8 International Journal of Infammation the standard condition. In the HFD alone and NAC-treated HFD groups a signifcant increase in the total expression of NF-κB was noticed (HFD: +73.3%, p < 0.0001, HFD + NAC: +30.5%, p < 0.0001, vs. Control group; Figure 5 . Te expression of selected proteins was examined by the western blot method. Te data are expressed as mean ± standard deviation (SD) and based on six independent determinations (n � 6). Te signifcant diferences were assessed by two-way ANOVA followed by a respective post hoc test (Tukey's test and t-test). * p < 0.05, signifcant diference compared to the control group; # p < 0.05, signifcant diference compared to the HFD group.
International Journal of Infammation 9

Efect of Eight-Week NAC Treatment on the Concentration of Oxidative Stress Parameters in the Left Ventricle Tissue of Rats Subjected to a Standard and a High-Fat Diets.
In the HFD alone group the concentration of SOD2 was substantially downgraded (HFD: −12.1%, p < 0.0001; Figure 6(a)) than in the control group. Whereas, in the HFD + NAC group the SOD2 level was no statistically different (HFD + NAC: p � 0.9361 and p � 0.0627, vs. Control and HFD groups, respectively; Figure 6(a)). Interestingly, in the HFD alone group we noticed a signifcant decrease in the level of CAT and GSH (CAT-HFD: −20.4%, p � 0.0293, vs. control group; Figure 6(b); GSH-HFD: −17.1%, p � 0.0035, vs. Control group; Figure 6(b)), which was restored by the chronic NAC administration (CAT − HFD + NAC: +31.1%, p � 0.0022, vs. Control group; Figure  6(b); GSH-HFD + NAC: +15.1%, p � 0.0031, vs. Control group; Figure 6(b)). Te preventive efect of NAC was disclosed as the increment in antioxidant capacity, i.e., increases in the content of CAT and GSH levels. Te lipid overload conditions disclosed a notable elevation in the concentration of 4-HNE (HFD: +61.9%, p � 0.0020, vs. control group; Figure 6(d)) whereby this change was abolished by NAC treatment (HFD + NAC: −25.3%, p � 0.0127, vs. HFD group; Figure 6(d)). Moreover, we found a pronounced increase in the amount of MDA in the HFD and HFD with NAC administration groups (HFD: +63.0%, p � 0.0001, HFD + NAC: +40.4%, p � 0.0022; Figure 6(e)) in comparison with the control subjects. In relation to the HFD group, the MDA concentration was remarkedly lower in rats fed a high-fat chow with NAC supplementation (HFD + NAC: −13.9%, p � 0.0078; Figure 6(e)). Te reduction of 4-HNE and MDA levels after application of NAC with HFD may be refect the protective efect on the deterioration of heart function caused by obesity. In all examined groups the concentration of AGE was appreciable changed (HFD: +41.3%, p � 0.0022, NAC: −22.0%, p � 0.0293, HFD + NAC: +37.2%, p < 0.0001; Figure 6

Discussion
Infammation is a concomitant factor in obesity-related cardiovascular diseases, contributing to the progression of myocardial fbrosis and loss of heart function [19]. Te regulation of the infammatory response is based on the control of the level and biological activity of mediators, commonly known as eicosanoids. We investigated the efect of N-acetylcysteine on AA derivatives as key parameters of infammation state and related parameters of oxidative balance in the left ventricle tissue of HFD-fed obese rats (the precise characterization of the obesity model in rats was previously described in our studies [20][21][22]). In our research, chronic oversupply of FA caused an increase in the myocardial AA content in each examined fraction, i.e., PL, TAG, DAG, and FFA. Similarly, an elevated concentration of AA in plasma PL, DAG, and FFA fractions was observed. Te increment of AA content in the various lipid pools suggests the development of infammation in the heart, which may deteriorate heart functioning [23]. In line with our observations, the results obtained by Pakiet et al. showed an increase in the content of n-6 PUFA, including AA, in the FFA and PL fractions in the plasma as well as in the heart tissue of HFD-treated mice [24]. Interestingly, we exhibited that NAC supplementation ameliorated an increase in myocardial arachidonic acid concentration induced by HFD-feeding in all examined fractions. We can suppose that the present reduction in AA concentration is the result of NAC supplementation revealing its anti-infammatory properties. Te AA pathway remains under the control of two crucial types of enzymes, i.e., cyclooxygenases catalyzed Table 3: Te concentration of selected cytokines and chemokines, i.e., granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-regulated oncogenes/keratinocyte chemoattractant (GRO/KC), interferon c (IFN-c), interleukin 1α (IL-1α), interleukin 1β (IL-1β), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 10 (IL-10), interleukin 12 p70 (IL-12 p70), interleukin 13 (IL-13), interleukin 17A (IL-17A), interleukin 18 (IL-18), monocyte chemoattractant protein 1 (MCP-1), macrophage infammatory protein 1α (MIP-1α), macrophage infammatory protein 3α (MIP-3α), regulated upon activation, normal T-cell expressed and secreted (RANTES), tumor necrosis factor α (TNF-α), and vascular endothelial growth factor (VEGF) after eight-week N-acetylcysteine (NAC) treatment in the left ventricle tissue of rats fed a standard diet (control) or a high-fat diet (HFD). Te cytokines and chemokines content was examined by the bio-plex immunoassay kit. Te data are expressed in picograms per milliliter as the mean ± standard deviation (SD) and based on ten independent determinations (n � 10). Te signifcant diferences were assessed by two-way ANOVA followed by a respective post hoc test (Tukey's test and t-test). * p < 0.05, signifcant diference compared to the control group; # p < 0.05, signifcant diference compared to the HFD group.

Parameter
Control HFD NAC HFD + NAC G-CSF   International Journal of Infammation the generation of 2-series prostaglandins and thromboxanes (TX), and also lipoxygenases (LOX), which participate in the formation of 4-series leukotrienes (LT) and lipoxins (LX) [25]. In our research, a high-fat diet resulted in elevated myocardial expression of both COX isoforms (COX-1 and COX-2), which mediates the production of prostanoids. Studies conducted on hypertensive rats revealed that animals receiving an HFD for 10-week had an increased COX-1 expression with no changes in COX-2 expression in the aorta [26]. On the other hand, in research conducted on mice fed an HFD for 8-week a rise in COX-2 expression in the myocardial tissue was reported [27]. Tis small discrepancy in COX-2 expression may be a result of diferent experimental materials-aorta and myocardium. COX-1 is constitutively secreted by most mammalian tissues and is called a "housekeeping" enzyme that regulates metabolism processes under physiological conditions [28]. COX-1 is mainly responsible for the production of thromboxane A2 (TXA2), but PGI2 is also responsible for maintaining cell integrity. In addition, PGI2 is considered a potent vasodilator and inhibits platelet aggregation [29,30]. Many studies have noticed that PGI2 attenuates cardiac hypertrophy and fbrosis through inhibited collagen synthesis [30,31]. On the other hand, COX-2 is an enzyme, which expression is revealed in response to the development of infammation state and might be enhanced by the release of proinfammatory cytokines [29]. Moreover, COX-2 regulates the frst step of AA conversion to proinfammatory PGE2 and also anti-infammatory PGI2 which contribute to the infammatory response [23]. Several fndings showed that PGE2 levels increase in cardiovascular diseases, e.g., cardiac hypertrophy or myocardial fbrosis [32,33]. In our study, we demonstrated that an enhanced expression of COX-2 resulted in a rise in PGE2 level, which was abolished by N-acetylcysteine supplementation. Our observations are consistent with Guo et al.' data in which 8-week NAC administration decreased the expression of COX-2 in the heart of diabetic rats [34]. NAC intensifed the infuence of common nonsteroidal anti-infammatory drugs (rofecoxib and diclofenac) by inhibiting COX-2 activity, resulting in a signifcant reduction in PGE2 level, which was observed by Hofer et al. on human monocytes with lipopolysaccharideinduced PGE2 formation [2]. Te above data show a decline in the content of PGE2 after N-acetylcysteine treatment, thus pointing to a potential role of NAC in infammationassociated cardiac obesity. As we mentioned, LOX is a second major enzyme family involved in the AA metabolism pathway, leading to the formation of anti-infammatory LX and proinfammatory LT [23]. In our study, we demonstrated an elevation in the total expression of 5-LOX after 8 weeks of high-fat feeding. As a consequence of the mentioned alteration in 5-LOX expression, the concentration of LTB4 and LTC4 increased. LTB4 is one of the crucial chemoattractant in the early phase of infammation that causes the infltration of neutrophils [35]. LTB4 also participates in the development of atherosclerosis and myocardial impairment [29]. In contrast, LTC4 is associated with the occurrence of oxidative stress and apoptosis in the myocardial tissue [36]. Becher et al. revealed that the incubation of cardiomyocytes with LTC4 led to an elevated generation of reactive oxygen species, resulting in the activation of the apoptosis process, which was refected in fragmented and/or pyknotic cell nuclei. Importantly, pharmacological inhibition of the of LTC4 receptor prevented ROS production and simultaneously attenuated cardiomyocyte apoptosis [36]. Te above efects prove that LTB4 and LTC4 weaken myocardial function. In our study, the administration of NAC abolished the increased 5-LOX expression, which resulted in a decline in the content of LTB4 and LTC4. Following our results, the study conducted by Karuppagounder et al. disclosed that NAC prevented against hemin-induced ferroptosis through scavenger proinfammatory lipid derivatives such as LTB4 and LTC4 generated by 5-LOX activity in nerve cells [37]. We can Te content of oxidative balance parameters was examined by the enzyme-linked immunosorbent assay (ELISA) and the colorimetric kits. Te data are expressed as mean ± standard deviation (SD) and based on ten independent determinations (n � 10). Te signifcant diferences were assessed by two-way ANOVA followed by a respective post hoc test (Tukey's test and t-test). * p < 0.05, signifcant diference compared to the control group; # p < 0.05, signifcant diference compared to the HFD group.
presume that N-acetylcysteine has a protective infuence on the infammation state in HFD-induced cardiac injury by ameliorating the expression of 5-LOX and the generation of 4-series leukotrienes. Herein, we also demonstrated a signifcant increase in the total expression of 12/15-LOX in cardiac tissue of HFD-treated rats. A previous study showed that 3-month of high-fat feeding prompted a rise in the expression of 12/15-LOX in the arteries of wild-type mice, causing the breakage of tight junctions and macrophage adhesion that underlie the mechanism of atherosclerosis [38]. Te data also suggested that an enhanced expression of 12/15-LOX in a late infammation state could result from an increased PGE2 level [23]. It is important to note that 12/15-LOX catalyzes the synthesis of 4-series lipoxins such as LXA4, which concentration in the HFD group was decreased. LXA4 is considered an atheroprotective factor due to the inhibition of proinfammatory cytokines generation and the prevention of neutrophil chemotaxis [23,29]. In the present study, chronic NAC treatment of rats fed an HFD enhanced the LXA4 level in the left ventricle, which may limit obesity-induced infammation by decreasing proinfammatory cytokines content [39]. We also demonstrated a decrease in the expression of Nrf-2 and Bcl-2 with simultaneous increases in the expression of NF-κB in the left ventricle of obese rats, which regulates the synthesis of infammatory cytokines and chemokines [40,41]. In light of these reports, we observed that in obese rats NAC ameliorated myocardial NF-κB expression, inhibiting the production of proinfammatory cytokines, such as TNF-α.
Another study conducted on a rabbit model with doxorubicin-induced heart failure showed that NAC, by increasing the total antioxidant capacity and reducing NF-κB activation, diminished cardiomyocyte apoptosis and the expression of proinfammatory 8-iso-prostaglandin F2α. Tis fnding suggests that NAC improves the structure and functioning of the myocardium under an infammation state [42]. So, it may be presumed that in the left ventricle of rats from the HFD + NAC group we observed a decrease in the total expression of NF-κB, which led to a weakness in the infammation processes by reducing the level of cytokines, i.e., IL-1α and TNF-α. What is more interesting, we also revealed a signifcant infuence of NAC on infammatory parameters in the cardiac tissue of rats fed a standard diet. In our research, the chronic administration of NAC to rats from the control group caused the diminishment of AA concentration in the DAG and FFA pools along with a decrease in the myocardial content of its proinfammatory eicosanoid derivatives, i.e., PGE2 and LTC4. According to existing literature, the impact of NAC on infammatory mediators in an animal model fed a standard diet is unclear. Following our results, studies conducted on astrocytes cultured under normoxic conditions showed that NAC reduced the release of arachidonic acid into the media, which can potentially be responsible for the development of infammation, and thus protected against the toxic efects of AA [1]. Concomitantly, we also observed the reduction in NF-κB expression in the left ventricle from the control rats treated with NAC. In line with this alteration, a decrease in the content of the following proinfammatory cytokines: IL-1α, IL-1β, MIP-1α, MIP-3α, RANTES, TNF-α was observed, limiting the occurrence of infammation. Te reduction of the above-mentioned parameters in the group of rats fed a standard chow indicates the benefcial role of NAC as an anti-infammatory compound preventing the development of heart dysfunction. It is known that oxidative stress is one of the important factors for cardiac damage incidence. Oxidative stress is defned as an imbalance between the antioxidant capacity and the generation of reactive oxygen species and free radicals [43]. Tere is some evidence showing a decrease in the content of enzymatic antioxidants-SOD2, CAT, and nonenzymatic antioxidants-GSH in CVD induced by highfat feeding [44,45]. In line with the above reports, in our study, the content of antioxidant markers, i.e., SOD2, CAT, and GSH, was decreased during feeding with HFD. It should be noted that reduced glutathione is a common antioxidant in the cardiovascular system that plays a major role in the inactivation of ROS or as a cofactor for glutathione peroxidase, causing the degradation of hydrogen peroxide [46]. Tus, GSH restores intracellular redox balance and also inhibits the inactivation of nitric oxide generated by the endothelium, altering vasomotor reactivity [43]. In agreement with our study, Andrich et al. revealed a decreased GSH level in the skeletal muscle of rats fed an HFD for 2 weeks [47]. In our study, NAC supplementation increased the concentration of GSH in rats fed an HFD. Te benefcial efects of NAC administration stem from the counteraction of ROS and the supplementation of a greater content of cysteine, which is a precursor for the most common antioxidant-GSH [46,48]. De Mattia et al. showed that NAC treatment caused an increase in GSH concentration and ameliorated the adhesion of molecules to the endothelium, ensuring its proper functioning [49]. In turn, catalase also exhibits action against cardiac oxidative injury by catalyzing the decomposition of hydrogen peroxide and inactivating this reactive form of oxygen into nontoxic products [50]. Mabrouki et al. demonstrated a reduction in CAT, and SOD levels in cardiac tissue within 12 weeks of HFD-treated rats [51]. In our study, an 8-week NAC application also restored the lowered level of CAT in rats receiving an HFD. Qin et al. demonstrated that catalase overexpression in a cardiomyopathy-transgenic mice model prevented against cardiac remodeling and its progression to heart failure [52]. In the herein study, we also noticed that feeding an HFD induced an increase in the concentration of lipid glycation and peroxidation products, i.e., 4-HNE, MDA, and AGE in the left ventricle tissue. Te research conducted by Hartog et al. revealed that AGE causes the formation of additional bonds called cross-links between extracellular matrix proteins (collagen, elastin, and laminin) decreasing their elasticity and promoting cardiac dysfunction [53]. Previous studies showed that increased oxidative stress results in the accumulation of AGE, which is associated with the development and progression of myocardial failure [54]. In the case of 4-HNE, it is a toxic lipid peroxidation product, which is formed in the reaction of ROS with cellular biomolecules, such as lipids, especially PUFA, leading to oxidative modifcations which further result in impairment in cellular International Journal of Infammation activities [55]. Another product of lipid peroxidation processes is MDA. In patients with coronary heart disease, the serum MDA concentration was increased with a concomitant increase in oxygen-free radicals, indicating the proatherogenic property of this oxidative damage marker [56]. In our study, we observed that alterations in the level of lipid peroxidation products, such as 4-HNE and MDA, induced by HFD were abolished by NAC treatment. Interestingly, Arstall et al.'s study established that NAC administration in combination with standard treatment (streptokinase and/or nitroglycerine) in patients with acute myocardial infarction signifcantly reduced oxidative stress via diminished MDA content and increased GSH concentration in plasma, resulting in improved left ventricle function [11]. Tese fndings imply that N-acetylcysteine protects cardiomyocytes from damage by decreasing lipid peroxidation products and enlarging the level of antioxidants thereby improving the function of cardiac muscle.
In our study, to obtain a homogeneous group, we used only male rats. Tere is a lot of evidence that gonadal hormones, especially estradiol in females alter lipid metabolism, leading to higher accumulation or conversion of AA from precursors compared to males [57]. Studies conducted by Lyman et al. showed that female rats maintained an increase content of AA in the plasma PL fraction than did males, pointing to the direct infuence of estradiol, as the main cause of this diference [58]. Tere is one limitation to this study, which does not compare both males and females causing impossible an accurate assessment of NAC efects of on HFD-induced obesity depending on gender. What is more, hyperphagia and weight gain are strongly correlated with sex. Studies demonstrated that male rats presented higher caloric intake and mass body gain caused by HFD compared to females. Further, a delay in HFD-induced obesity and the occurrence of metabolic disturbances due to a lower level of hyperphagia and higher energy expenditure in female rats were observed. Interestingly, female rats are more active than males, resulting in lesser weight gain, which could be one of the possible factors for the obesity resistance in females [59].

Conclusion
Obesity is closely related to higher circulating FFA concentrations, which promote ectopic lipid accumulation, thereby adversely afecting cellular structure and functions. Tese changes lead to the development of infammation state and oxidative stress, which are important factors for cardiovascular disease occurrence. In this study, we established that NAC supplementation might be a potential agent for preventing the occurrence of infammation in obesityrelated cardiac diseases by a diminishment in the AA concentration, especially in the phospholipid fraction. Nacetylcysteine administration reduced the expression of COX-2, leading to a decrease in the content of proinfammatory PGE2, and also reduced the expression of 5-  LOX, resulting in a decrease in the concentration of leukotrienes, namely LTB4 and LTC4. Noteworthy, the observed reduction in the NF-κB expression after NAC supplementation weakened infammatory signaling via a decline in the content of proinfammatory cytokines. NAC also ameliorated myocardial oxidative stress in rats fed an HFD through an increase in antioxidant content, especially GSH and CAT, with simultaneously a decrease in the level of lipid peroxidation products-4-HNE and MDA. Based on the presented results (Scheme 1) it can be concluded that N-acetylcysteine has a great potential to protect against the development of myocardial infammation and oxidative stress in rats with obesity induced by a high-fat diet.